20210601/0630_SS2_ZY, final

ILC2 IL25+/-, in vitro (02/)04/48/72h
(02h invitro ILC2 not stable, not involved in merged downstream analysis)

load processed data

TPM matrix

matt_raw <- read.table("./matrix/RNAseq.SS2_ZY_20210701.raw_tpm.gene.csv", header = T, sep = ",")
rownames(matt_raw) <- matt_raw$gene
matt_raw <- matt_raw[,2:ncol(matt_raw)]

matt_pc <- read.table("./matrix/RNAseq.SS2_ZY_20210701.filt_tpm.pc_gene.csv", header = T, sep = ",")
rownames(matt_pc) <- matt_pc$gene
matt_pc <- matt_pc[,2:ncol(matt_pc)]

idx.C04h <- c(13:16,9:12)
idx.C48h <- c(21:24,17:20)
idx.C72h <- c(29:32,25:28)

idx.IL25_C48hvsC04h <- c(21:24,13:16)
idx.IL25_C72hvsC04h <- c(29:32,13:16)
idx.IL25_C72hvsC48h <- c(29:32,21:24)

idx.nonIL25_C48hvsC04h <- c(17:20,9:12)
idx.nonIL25_C72hvsC04h <- c(25:28,9:12)
idx.nonIL25_C72hvsC48h <- c(25:28,17:20)
dim(matt_raw)
## [1] 55413    32
head(matt_raw)
##               C02h1 C02h2 C02h3 C02h4 C02h5 C02h6 C02h7 C02h8 C04h1 C04h2 C04h3
## 0610005C13Rik  0.00  0.00  0.83  0.77  0.00  0.19  0.59  0.28  0.16  0.86  0.68
## 0610006L08Rik  0.00  0.51  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00
## 0610009B22Rik 23.95 13.10 14.83 15.26 23.85 18.14 18.32 26.16 21.26 24.08 24.88
## 0610009E02Rik  3.32  0.50  0.00  5.02  0.65  0.00  2.90  0.00  0.97  0.00  0.37
## 0610009L18Rik  0.00  2.29  8.58  0.00  1.24  3.05  0.92  0.74  1.85  0.86  0.00
## 0610010F05Rik  0.62  2.52  2.67  2.36  2.69  3.58  4.11  2.97  3.59  4.49  5.33
##               C04h4 C04h5 C04h6 C04h7 C04h8 C48h1 C48h2 C48h3 C48h4 C48h5 C48h6
## 0610005C13Rik  0.48  0.78  0.79  0.13  0.00  0.09  0.10  0.08  0.48  0.66  0.41
## 0610006L08Rik  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00
## 0610009B22Rik 20.31 14.88 16.94 21.17 10.63 41.41 31.91 41.36 36.33 33.20 32.68
## 0610009E02Rik  0.69  0.67  0.00  0.00  0.00  0.00  1.14  0.00  2.06  0.00  0.68
## 0610009L18Rik  2.81  0.63  0.63  0.00  1.33  3.11  1.64  4.86  2.96  1.78  0.55
## 0610010F05Rik  6.08  5.10  5.59  3.20  4.13  3.08  3.56  4.39  5.36  5.30  6.01
##               C48h7 C48h8 C72h1 C72h2 C72h3 C72h4 C72h5 C72h6 C72h7 C72h8
## 0610005C13Rik  0.27  0.76  0.00  0.16  0.00  0.43  0.36  0.00  0.00  0.22
## 0610006L08Rik  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00  0.00
## 0610009B22Rik 37.04 32.29 33.17 44.37 39.30 35.51 25.00 31.87 32.94 29.36
## 0610009E02Rik  0.00  0.69  0.00  0.45  1.42  0.00  0.00  0.87  0.00  0.71
## 0610009L18Rik  1.37  1.51  0.51  1.28  1.80  2.01  1.92  0.00  3.11  3.27
## 0610010F05Rik  4.15  4.98  2.18  3.66  2.98  6.13  3.47  3.29  2.50  4.06
dim(matt_pc)
## [1] 10799    32
head(matt_pc)
##               C02h1 C02h2 C02h3 C02h4 C02h5 C02h6 C02h7 C02h8 C04h1 C04h2 C04h3
## 0610009B22Rik 23.95 13.10 14.83 15.26 23.85 18.14 18.32 26.16 21.26 24.08 24.88
## 0610010F05Rik  0.62  2.52  2.67  2.36  2.69  3.58  4.11  2.97  3.59  4.49  5.33
## 0610010K14Rik 46.84 35.70 36.72 38.10 36.77 38.81 43.01 27.85 51.61 66.46 67.06
## 0610012G03Rik 44.66 40.21 31.76 26.73 29.16 28.98 27.51 38.09 45.28 40.28 33.81
## 0610030E20Rik 22.03 27.53 35.26 28.45 27.64 31.41 29.62 31.91 26.32 21.54 20.28
## 1110002E22Rik  0.00  0.30  0.10  0.00  0.55  0.41  0.54  0.32  0.22  0.23  0.25
##               C04h4 C04h5 C04h6 C04h7 C04h8  C48h1  C48h2 C48h3 C48h4 C48h5
## 0610009B22Rik 20.31 14.88 16.94 21.17 10.63  41.41  31.91 41.36 36.33 33.20
## 0610010F05Rik  6.08  5.10  5.59  3.20  4.13   3.08   3.56  4.39  5.36  5.30
## 0610010K14Rik 61.19 58.70 68.08 66.62 55.93 104.24 113.16 90.76 93.41 75.29
## 0610012G03Rik 44.32 36.24 32.39 34.34 33.24  74.02  67.13 60.04 63.21 48.69
## 0610030E20Rik 22.55 25.56 19.27 19.53 22.83  15.62  20.53 20.98 21.39 10.88
## 1110002E22Rik  0.25  0.95  0.75  0.81  0.92   0.00   0.02  0.00  0.02  0.00
##               C48h6 C48h7 C48h8 C72h1 C72h2 C72h3 C72h4 C72h5 C72h6 C72h7 C72h8
## 0610009B22Rik 32.68 37.04 32.29 33.17 44.37 39.30 35.51 25.00 31.87 32.94 29.36
## 0610010F05Rik  6.01  4.15  4.98  2.18  3.66  2.98  6.13  3.47  3.29  2.50  4.06
## 0610010K14Rik 83.29 86.47 76.81 76.53 90.27 90.21 80.46 47.72 74.76 66.51 63.84
## 0610012G03Rik 59.05 59.43 49.57 70.56 83.64 66.06 62.72 76.64 61.32 62.50 67.08
## 0610030E20Rik 10.61 11.35 10.58 18.00 23.29 19.15 24.96 11.92 12.06 12.79  9.80
## 1110002E22Rik  0.00  0.02  0.00  0.00  0.04  0.00  0.00  0.00  0.00  0.00  0.00

clustering

PCA

summary of PCs:
## Importance of components:
##                            PC1     PC2     PC3      PC4     PC5     PC6     PC7
## Standard deviation     29.9929 21.2282 16.9714 10.24773 7.40695 6.56617 4.08178
## Proportion of Variance  0.4498  0.2253  0.1440  0.05251 0.02743 0.02156 0.00833
## Cumulative Proportion   0.4498  0.6751  0.8191  0.87163 0.89906 0.92062 0.92895
##                           PC8    PC9    PC10    PC11    PC12    PC13    PC14
## Standard deviation     3.8207 3.6874 3.62840 3.36029 3.06920 2.87807 2.71365
## Proportion of Variance 0.0073 0.0068 0.00658 0.00565 0.00471 0.00414 0.00368
## Cumulative Proportion  0.9363 0.9430 0.94963 0.95527 0.95998 0.96412 0.96781
##                           PC15    PC16    PC17    PC18    PC19    PC20    PC21
## Standard deviation     2.44441 2.35167 2.31266 2.20761 2.15059 2.05765 1.99155
## Proportion of Variance 0.00299 0.00277 0.00267 0.00244 0.00231 0.00212 0.00198
## Cumulative Proportion  0.97079 0.97356 0.97623 0.97867 0.98098 0.98310 0.98508
##                           PC22    PC23    PC24    PC25    PC26    PC27    PC28
## Standard deviation     1.98331 1.91795 1.82857 1.79572 1.73851 1.67774 1.63592
## Proportion of Variance 0.00197 0.00184 0.00167 0.00161 0.00151 0.00141 0.00134
## Cumulative Proportion  0.98705 0.98889 0.99056 0.99217 0.99368 0.99509 0.99643
##                           PC29   PC30    PC31    PC32
## Standard deviation     1.57397 1.5495 1.50452 1.7e-14
## Proportion of Variance 0.00124 0.0012 0.00113 0.0e+00
## Cumulative Proportion  0.99767 0.9989 1.00000 1.0e+00

tSNE

correlation of all filtered protein-coding genes, grouping by correlation

clustering (without C02h)

PCA

summary of PCs:
## Importance of components:
##                            PC1     PC2      PC3     PC4     PC5     PC6     PC7
## Standard deviation     33.0232 21.6324 12.76393 8.80483 6.92260 4.39204 4.26366
## Proportion of Variance  0.5453  0.2340  0.08146 0.03876 0.02396 0.00964 0.00909
## Cumulative Proportion   0.5453  0.7792  0.86070 0.89947 0.92343 0.93307 0.94216
##                            PC8     PC9    PC10    PC11    PC12   PC13    PC14
## Standard deviation     4.16336 3.86310 3.09133 2.94217 2.84744 2.6449 2.59506
## Proportion of Variance 0.00867 0.00746 0.00478 0.00433 0.00405 0.0035 0.00337
## Cumulative Proportion  0.95083 0.95829 0.96307 0.96740 0.97145 0.9749 0.97832
##                           PC15    PC16    PC17    PC18    PC19    PC20   PC21
## Standard deviation     2.51401 2.37934 2.30841 2.24594 2.18831 2.07612 2.0488
## Proportion of Variance 0.00316 0.00283 0.00266 0.00252 0.00239 0.00216 0.0021
## Cumulative Proportion  0.98148 0.98431 0.98697 0.98949 0.99189 0.99404 0.9961
##                           PC22    PC23     PC24
## Standard deviation     2.00466 1.92337 1.08e-14
## Proportion of Variance 0.00201 0.00185 0.00e+00
## Cumulative Proportion  0.99815 1.00000 1.00e+00

tSNE

correlation of all filtered protein-coding genes, grouping by correlation

DEGs

mark_rowname <- function(DEGs){
  rownames(DEGs) <- DEGs$gene
  return(DEGs)
}


DEGs.C04h <- mark_rowname(read.table("./DEGs/IL25_vs_nonIL25/edgeR_DEGs_C04h5.6.7.8_vs_C04h1.2.3.4.csv",
                        header = T, sep = ","))
DEGs.C48h <- mark_rowname(read.table("./DEGs/IL25_vs_nonIL25/edgeR_DEGs_C48h5.6.7.8_vs_C48h1.2.3.4.csv",
                        header = T, sep = ","))
DEGs.C72h <- mark_rowname(read.table("./DEGs/IL25_vs_nonIL25/edgeR_DEGs_C72h5.6.7.8_vs_C72h1.2.3.4.csv",
                        header = T, sep = ","))

DEGs.IL25_C48hvsC04h <- mark_rowname(read.table("./DEGs/times_IL25/edgeR_DEGs_C48h5.6.7.8_vs_C04h5.6.7.8.csv",
                        header = T, sep = ","))
DEGs.IL25_C72hvsC04h <- mark_rowname(read.table("./DEGs/times_IL25/edgeR_DEGs_C72h5.6.7.8_vs_C04h5.6.7.8.csv",
                        header = T, sep = ","))
DEGs.IL25_C72hvsC48h <- mark_rowname(read.table("./DEGs/times_IL25/edgeR_DEGs_C72h5.6.7.8_vs_C48h5.6.7.8.csv",
                        header = T, sep = ","))

DEGs.nonIL25_C48hvsC04h <- mark_rowname(read.table("./DEGs/times_nonIL25/edgeR_DEGs_C48h1.2.3.4_vs_C04h1.2.3.4.csv",
                        header = T, sep = ","))
DEGs.nonIL25_C72hvsC04h <- mark_rowname(read.table("./DEGs/times_nonIL25/edgeR_DEGs_C72h1.2.3.4_vs_C04h1.2.3.4.csv",
                        header = T, sep = ","))
DEGs.nonIL25_C72hvsC48h <- mark_rowname(read.table("./DEGs/times_nonIL25/edgeR_DEGs_C72h1.2.3.4_vs_C48h1.2.3.4.csv",
                        header = T, sep = ","))

C04h: IL25 vs nonIL25

using cutoff: p.adjust <0.05, FC >1.5
## C04h: IL25 vs nonIL25  
## 
## DEGs:  2139 
##   up:  807 
## down:  1332

## Warning: The input is a data frame, convert it to the matrix.

## Warning: The input is a data frame, convert it to the matrix.

C48h: IL25 vs nonIL25

using cutoff: p.adjust <0.05, FC >1.5
## C48h: IL25 vs nonIL25  
## 
## DEGs:  3163 
##   up:  1530 
## down:  1633

## Warning: The input is a data frame, convert it to the matrix.

## Warning: The input is a data frame, convert it to the matrix.

C72h: IL25 vs nonIL25

using cutoff: p.adjust <0.05, FC >1.5
## C72h: IL25 vs nonIL25  
## 
## DEGs:  2602 
##   up:  1269 
## down:  1333

## Warning: The input is a data frame, convert it to the matrix.

## Warning: The input is a data frame, convert it to the matrix.

IL25: C48h vs C04h

using cutoff: p.adjust <0.01, FC >2
## IL25: C48h vs C04h  
## 
## DEGs:  3633 
##   up:  1925 
## down:  1708

IL25: C72h vs C04h

using cutoff: p.adjust <0.01, FC >2
## IL25: C72h vs C04h  
## 
## DEGs:  4093 
##   up:  2218 
## down:  1875

IL25: C72h vs C48h

using cutoff: p.adjust <0.05, FC >1.5
## IL25: C72h vs C48h  
## 
## DEGs:  1436 
##   up:  785 
## down:  651

nonIL25: C48h vs C04h

using cutoff: p.adjust <0.01, FC >2
## nonIL25: C48h vs C04h  
## 
## DEGs:  2352 
##   up:  1310 
## down:  1042

nonIL25: C72h vs C04h

using cutoff: p.adjust <0.01, FC >2
## nonIL25: C72h vs C04h  
## 
## DEGs:  2423 
##   up:  1305 
## down:  1118

nonIL25: C72h vs C48h

using cutoff: p.adjust <0.05, FC >1.2
## nonIL25: C72h vs C48h  
## 
## DEGs:  60 
##   up:  33 
## down:  27